c-Met is a 190 kD tyrosine kinase receptor made up of an extracellular α-chain which is linked by a disulphide bond to a transmembrane β-chain. c-Met is synthesised as a 170 kD single polypeptide that is proteolytically cleaved to form the α-chain and the β-chain. The mature α-chain is 45 kD and constitutes part of the sema domain. The sema domain is a conserved domain shared by semaphorins and plexins. This domain adopts a seven-bladed beta-propeller structure which is important for homo-dimerisation. In c-Met, both the α-chain and the β-chain form the sema domain that is necessary and sufficient for receptor dimerisation and ligand binding.
Hepatocyte growth factor (HGF) is the only known c-Met ligand. Upon HGF binding, c-Met receptor dimerises on the cell surface which results in autophosphorylation of tyrosine residues in the kinase domain. Autophosphorylation is thought to induce a conformational change in c-Met, exposing the docking site in the carboxyl-terminal tail of c-Met. This results in transphosphorylation of tyrosine residues in c-Met docking site. The docking site becomes available for recruitment of adaptor and signalling molecules resulting in the activation of various signalling pathways including the AKT/PI3K, RAS/MAPK and STAT pathways.
Aberrant c-Met activation of c-Met signalling pathways correlates with hyperproliferation, tumour cell invasion, tumour angiogenesis and poor prognosis in various human cancers. In addition, c-Met signalling protects the tumour cell by inhibiting apoptosis and inducing resistance towards cancer therapy, thus hampering the efforts of tumour treatment. c-Met as a cancer prognosis marker and its involvement in cancer metastasis and drug resistance makes c-Met a very attractive drug target.
Various mechanisms have been used to inhibit c-Met activation. Small molecule kinase inhibitors such as the PHA-665752, AM7 and SU11274 were extremely successful in inhibiting c-Met activation. However, toxicity issues due to off-target effects of the small molecule inhibitors are of major concern. In addition, SU11274 was reported to be ineffective against specific c-Met mutations.
The use of U1snRNA/ribozyme has also been reported to downregulate the expression levels of c-Met/HGF. However this method is not feasible for cancer treatment due to drug delivery issues. U1snRNA/ribozyme has to be efficiently delivered into every tumour cell to be expressed in order to be effective.
The use of HGF and c-Met fragments, such as NK4 and decoy-Met respectively, to compete for Met-HGF interactions has been examined. These competitive inhibitors show efficient inhibition of c-Met activation in vivo xenograft models; however, their clinical utility has yet to be determined.
Many antibodies, such as Herceptin (clinically known as Trastuzamab), have been clinically successful. Herceptin is a chimeric antibody targeted against the tyrosine receptor kinase HER2, used for breast cancer treatment.
With the success of therapeutic antibodies, attempts have been made to develop therapeutic antibodies against the Met-HGF axis. Neutralising antibodies targeted against HGF aimed to block Met-HGF interaction were developed. c-Met binding to HGF was only blocked when a combination of two or three different anti-HGF antibodies were used. Five fully human anti-HGF antibodies targeted against the β-chain of HGF have been developed. These antibodies were successful in blocking Met-HGF interaction in U-87MG glioblastoma cells.
Developing therapeutic bivalent antibodies targeted against c-Met has been challenging. Two monoclonal antibodies (DO-24 and DN-30) against the extracellular domain of c-Met have previously been developed. Interestingly, both monoclonal antibodies act as an agonist rather than an antagonist and activate c-Met signalling in vivo. To avoid c-Met activation by bivalent monoclonal antibodies, a DN-30 Fab fragment was engineered. DN-30 Fab retained its high binding affinity towards c-Met but lost its agonist activity towards c-Met. DN-30 Fab efficiently inhibited c-Met signalling by causing c-Met ectodomain shedding and receptor down regulation.
The one-arm 5D5 antibody (MetMab or clinically known as Onartuzumab) is a monovalent chimeric antibody targeted against c-Met. Like DN-30, bivalent 5D5 antibody became an antagonist when converted to a monovalent Fab. In contrast to Fab DN-30, MetMab acts as an antagonist by competing with HGF for c-Met binding and causes c-Met internalisation and down-regulation.
Thus, there is a need to provide new antagonist antibodies binding to c-Met that overcome, or at least ameliorate, one or more of the disadvantages described above.